Industry allocated project PHI allocated project

number number

FINAL REPORT 2017

AGRICULTURAL RESEARCH COUNCIL

PLANT PROTECTION RESEARCH INSTITUTE
P/Bag X134, Queenswood, Pretoria 0121

FINAL REPORT
Ref: PPRI-11/19

Improved virus detection and identification for the Wine

Grape Certification Scheme

Prepared by: Prof. G Pietersen

Tel: 012 420 3265 or 082 647 5326
Email: gerhard.pietersen@up.ac.za

Prepared for: Client: Winetech

Contact person: Anel Andrag
Tel: (021) 276 0499 Fax: 086 611 7817
Email: AndragA@winetech.co.za

JULY 2017

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SATI CFPA SAAPPA/SASPA DFTS Winetech

tarryn@satgi.co.za inmaak@mweb.co.za theresa@hortgro.co.za dappies@dtd.co.za andraga@winetech.co.za

Tel: 021 863-0366 Tel: 021 872-1501 Tel: 021 882-8470 Tel: 021 870 2900 Tel: 021 276 0499
X

FINAL REPORT
(2017)

1. PROGRAMME AND PROJECT LEADER INFORMATION

Research
Organisation Research Team Manager Project leader
Programme leader
Title, Dr. Isabel Rong Prof. Gerhard Pietersen Prof. Gerhard Pietersen
initials,
surname
Present Programme Manager Specialist Scientist and Extra- Specialist Scientist and
position Ordinary Professor Extra-Ordinary Professor
Address ARC-PPRI ARC-PPRI, c/o Dept. of ARC-PPRI, c/o Dept. of
Private bag X134 Microbiology and Plant Microbiology and Plant
Queenswood Pathology, University of Pretoria, Pathology, University of
0121 0002 Pretoria, 0002
Tel. / 012 808-8000 012 420 3265/0826475326 012 420
Cell no. 3265/0826475326
Fax 012 420 3266 012 420 3266
E-mail RongeI@arc.agric.za gerhard.pietersen@up.ac.za gerhard.pietersen@up.ac.za
Co-worker Student
Title, Dr. AEC Jooste Jennifer Wayland
initials,
surname
Present Researcher MSc student
position
Address ARC-PPR Dept. of Microbiology & Plant
Private bag X134 Pathology, University of
Queenswood, 0121 Pretoria, Pretoria, 0002
Tel. / 0128088197 012 420-3279
Cell no.
Fax 0128088299 012 420 3266
E-mail JoosteE@arc.agric.za Jennifer.Wayland@fabi.up.ac.za

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2. PROJECT INFORMATION

Research Organisation ARC-PPRI P

Project number (PPRI 11/19)
Project title Improved virus detection and identification for the Wine Grape
certification scheme
Short title Grapevine virus detection by NGS

Fruit kind(s) Wine grapes

Start date (mm/yyyy) 01-04-2012 End date (mm/yyyy) 31-03-2017

Key words Grapevine leafroll disease, technology transfer, presentations

Approved by Research Organisation Programme leader (tick box) X

3. EXECUTIVE SUMMARY

The South African Wine Grape Certification Scheme only tests for the most important of the
total range of viruses infecting Vitis worldwide. In this project, we intended expanding the
number of viruses that can be tested within the certification scheme. We developed a protocol in
which vines can be tested via multiple parallel PCR systems, each capable of detecting a range
of viral species within a specific genus, along with some virus specific systems. Should a
requirement exist for the identity of the individual virus species to be determined from genus
wide PCR system this will be done by Illumina sequencing of pooled preparations of the
amplicons from the PCR’s of any given vine, and by doing a number of such vines in parallel by
indexing such amplicon pools separately. We have implemented PCR to the following genera to
achieve this: Tombusviruses, Maculaviruses, Marafiviruses, Geminiviruses, Reoviruses,
Nepoviruses (clades A, B, and C), Vitiviruses, Foveaviruses, Trichoviruses, Vealriviruses,
Ampeloviruses, Closteroviruses, Ilarviruses, Alfamoviruses, Cucumoviruses, Potexviruses,
Badnaviruses, Potyviruses, Tobamoviruses and Luteoviruses. While the protocol was not be
evaluated against all viruses of grapevines, it is theoretically capable of detecting 41 viruses
reported to infect this host. Because of the unexpected phenomenon of index-leaching during
Illumina sequencing, it was necessary to develop a method of determining the positive/negative
threshold for the systems along with a protocol for read analysis. We tested the protocol
successfully on a number of vines from nuclear material to symptomatic field-collected material.
The protocol and appropriate reagents was transferred to the Virus diagnostic unit of the ARC-
PPRI for routine testing of viruses of grapevines. It is anticipated that utilisation of this system
by the Wine industry to test nuclear material, will lead to the production of vine planting material
of the highest virus-free status in the world, and will have a positive impact on South African
winegrape production which unfortunately will be difficult to quantify.

4. PROBLEM IDENTIFICATION AND OBJECTIVES

.

Within the South African Wine Grape Certification Scheme, only few of the total range of viruses
infecting Vitis worldwide, are tested for in nuclear-, foundation- and mother-block planting
material. In addition, a number of virus-like diseases of unknown aetiology are also tested using
biological indexing. In this project, we intend expanding the number of tests that could be used
in the certification scheme on primarily nuclear plants. To achieve this we will:

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1) Assess the ability of massively parallel sequencing (MPS) also known as next generation
sequencing (NGS) to serve as a means of improving routine virus detection within the South
African Wine Grape Certification Scheme of nuclear material in a non-virus-specific way for
known and unknown viruses of Vitis.
2) Assess the usefulness of MPS/NGS to replace IEM and PCR tests that are currently
outsourced at great cost to the industry,
3) Assess the use of MPS/NGS on highly multiplexed samples for the detection of viruses in
foundation block and mother-block planting material for viruses other than those associated with
leafroll.

As a second aspect, we intended evaluation of routine application of GLRaV-3 LAMP,

developed in project PPRI-GP2, and comparison with industry performed ELISA tests (as a
finalisation project PPRI-GP2), to detect GLRaV-3 in rootstocks, and to assess the usefulness
of a published LAMP protocol for the detection of Aster yellows phytoplasma in the field

5. WORKPLAN (MATERIALS AND METHODS)

In this project a protocol for routine, non-specific detection of viruses in Vitis plants was
developed using massively parallel sequencing and multiplexing to replace current IEM and
PCR (and possibly indexing) -based techniques done within the industry. To achieve this, the
following objectives were pursued.

1) Initially a protocol was developed as a proof-of concept study using six genus wide
primer pairs in RT-PCR systems to amplify members of the Clostero-, Viti-, Fovea- ,
GLRaV-4 like and Nepoviruses (two primer sets).

2) Various polymerase, reverse transcriptase, and buffer systems were evaluated in PCR
to obtain a standardised system that could be performed with multiple primer systems.

3) Using this, a known, multiple virus infected vine, which had been characterised in the
past (Black Spanish; PPRI Accession 90-0246) was tested for the presence of the viral
genera mentioned. Amplicons obtained were pooled and subjected to Illumina
sequencing in order to identify the viruses present.

4) In subsequent years additional primer sets directed at other viral genera were assessed
for their usefulness in parallel with the previously established RT-PCR systems. Some
DNA virus systems were also included for use in PCR.

5) Methods of data analysis of the Illumina reads obtained against the amplicons of the
PCRs tests were developed to streamline the search for virus within the Vitis host
background.

6) The multiplexing of samples was evaluated using indexed pools of cloned amplicons as
templates, in order to determine the number of samples that can be sequenced in
parallel (a critical component in reducing costs), without compromising the coverage of
the sequence data and accuracy of sequence identification.

7) Using the established template or template preparations and multiplexing conditions a

number of nuclear plants from the certification scheme were be selected and the virus
status determined by MPS.

8) Symptomatic vines from foundation or mother-block vineyards were analysed using the
developed MPS protocol.

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In the second aspect of the project (primarily a finalisation of project PPRI-GP2), we evaluated
1) the routine application of GLRaV-3 LAMP, developed in project PPRI-GP2, and comparison
with industry performed ELISA tests. (Finalisation project PPRI-GP2)

2) the GLRaV-3 LAMP developed in project PPRI-GP2, for the detection of GLRaV-3 in
rootstocks.

And, 3) the usefulness of a published LAMP protocol for the detection of Aster yellows
phytoplasma in the field.

6. RESULTS AND DISCUSSION

We searched the literature for viruses infecting grapevines worldwide. Lists were prepared
according to their respective genus or family. Published degenerate or universal (genus-wide)
PCR primers were identified and synthesized locally. Grapevine plants were identified within the
virus collection of PPRI that could serve as positive controls in the various PCR tests.
Collaborators worldwide were approached to provide appropriate positive controls where none
were available locally. Initially PCR’s were established to detect Closteroviruses,
Ampeloviruses, Velariviruses, Vitiviruses, Foveaviruses Trichoviruses, and Nepoviruses of
grapevines.

A generic (with regards PCR buffers and reagents barring the primers) RT-PCR protocol based
on GoScript reverse transcriptase and Gotaq polymerase enzymes, was developed in order to
standardise an amplification protocol for the different primer sets. The various PCR systems
were utilised within the generic master mix, in a proof of concept test on a single grapevine
plant previously shown to harbour multiple viruses. Amplicons obtained were pooled and
subjected to Illumina sequencing in order to identify the individual viruses obtained. Illumina
reads obtained were analysed for virus identity. The results confirmed the presence of
grapevine leafroll associated virus 1 (GLRaV-1), GLRaV-2, GLRaV-3 and grapevine fleck virus
(GFkV) all previously found in the vine using immune electron microscopy (ISEM). The plant
however also contained GLRaV-4, GLRaV-6, grapevine virus A (GVA), grapevine virus F (GVF)
and Rupestris stem pitting associated virus (RSPaV) as determined by using the polyspecific
PCR and Illumina sequencing protocol. At that stage grapevine virus F had not previously been
reported in South Africa nor had it been reported to be detectable with the specific primers
utilised. This confirmed the undoubted potential of the technique for the routine detection and
identification of multiple viruses of grapevines (either as mixed- or single infections), including
some related non-target viruses, within the certification scheme.

To assess the usefulness of tagging/indexing different samples for parallel sequencing it was
utilised to detect members of the viral genera listed above in a number of grapevine samples.
During these tests we obtained some false positives suggestive of index leaching or cross
sample contamination during the protocol. This required a considerable re-prioritisation of the
project to identify the steps at which contamination occurs, to minimize this, and to account for
this through the establishment of positive/negative thresholds in the analysis of the data.

Regions cognate to the amplicons from the PCR’s were cloned to serve as positive controls for
all systems developed and were used to create defined templates in order to establish an
analysis pipeline for Illumina data interpretation (Figure 1). During this process we assessed
the:
1) variability in results obtained, introduced by various steps of the protocol;
2) the limitations that pooling a number of numbers of amplicons from different tests to serve as
NGS template may have on the ability to detect each of the systems;

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3) effect of varying amounts of amplicon templates from individual tests on reads obtained; 4)
the effect that amplicon G:C ratio and amplicon length on the NGS reads obtained,
5) effect introduced by primer degeneracy, and
6) requirement of de novo assembly for detection of novel viruses.
The detailed establishment and requirements of this pipeline can be found in the 2015 and 2016
Progress reports of this project, but are summarized in Figure 1.

The number of PCR systems were increased during the course of the project to detect an
increasing number of virus genera (Table 1). In some instances, the importance of specific
viruses or the poor performance of polyspecific PCR’s, resulted in virus specific PCR’s being
utilised rather than genus-wide PCR’s (eg. GLRaV-1, -2 and -3).

The usefulness of the optimised protocol was assessed on nuclear material and some field-
grown material with abnormalities submitted by Mr. T. Oosthuizen (Vititec) and Mrs. M. Louw
(Bosman Brothers).

The samples from Vititec were also sent to Waite Diagnositic for parallel testing in order to
validate those obtained at UP/PPRI. The samples tested negative for the majority of the PCR
systems (Table 5 of 2016 Progress report). Four samples yielded very feint bands in the
GLRaV-3 PCR possibly due to the presence of a GLRaV-3 variant or low concentration of
GLRaV-3 or from a related, cross-reacting virus. Many samples analysed yielded feint amplicon
bands within the Viti-Fovea virus PCR, and in two instances these bands were clear. It is
evident that further studies on this group of viruses needs to be performed.

A total of 56 wine grape samples were collected from the regions of Somerset Wes and
Stellenbosch in the Western Cape, including samples of Cabernet Sauvignon, Affenthaler,
Bacchus, Aleatico nero, Capes Donnes Seedling, Blaue Kadarka, Cabernet franc, Grand noir
de la Calmette, Majestic, Merlot, Malbec and Shiraz. The wine grape samples were selected
based on various virus-like abnormalities or particularly severe leafroll symptoms.

With additional THRIP funding, a total of 49 table grape samples (table grapes selected as they
would have a greater likelihood of “unusual” viruses) were collected from the Western Cape in
the regions Hex river valley, Paarl and Wellington. Included were Vitis vinifera cv. Starlight,
Crimson, La Rochelle, Red Globe, Prime, Melody, Autumn Royal, Sugra 19 and Waltham cross.
The samples were selected based on virus like disease symptoms. All of these samples were
subjected to virus analysis using the expanded grapevine virus PCR protocol (Table 2, 2016
Progress report).

In a previous project (GP2) RT-LAMP was shown to be a viable alternative to ELISA for the
detection of GLRaV-3 on site, however actual implementation in these environments were
problematic due to either contamination of the LAMP reactions or to failure of the reaction when
utilised by industry. In the current project we finalised the optimisation and assessment of the
LAMP for the detection of GLRaV-3 in white cultivars and rootstocks in the laboratory and
published this work. Attempts to modify the protocol for field use was not very successful due to
the unavailability of suitable sealable reagent vessels, and as the desired reporter dye
“Genefinder” (red/green colour change) could not be accessed from China. We intend using a
microcrystalline wax-dye capsule according to Tao et al. (2011) to detect GLRaV-3 in rootstock
samples in a recently approved Winetech project on rootstocks as a means of preventing the
contamination n problems inherent in our previous protocol.

The ability of RT-LAMP to detect GLRaV-3 in grapevine rootstocks was confirmed when 78% of
samples of five different rootstocks (R99, R110, 101-14, SC, Ruggeri) inoculated with GLRaV-3
tested positive for GLRaV-3 using this method. ELISA on the same rootstocks had only
detected GLRaV-3 in 28%. The lowest incidence of RT-LAMP detection was with R110 where 3

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of 9 samples tested were negative while the highest was with Salt Creek where only one of 11
samples tested was negative.

Detection of Aster yellows was successful against AY-infected grapevines as well as Mgenia
fuscovaria individuals using the LAMP protocol of Tomlinson et al., 2010.

7. COMPLETE THE FOLLOWING TABLE

Target
Extension Date
Milestone Date Achievement
Date completed
1. Develop 04-2013 Use six genus wide primer pairs
multiple genus- in RT-PCR systems to amplify
wide PCR’s members of the Clostero-, Viti-,
and NGS Fovea- , GLRaV-4 like and
protocol as a 04-2013 Nepoviruses (two primer sets)
proof-of
concept study.

04-2013 RT-PCR protocol based on

2. Standardise GoScript reverse transcriptase
polymerase, and Gotaq polymerase enzymes
reverse developed for use in multiple
transcriptase, 04-2013 PCR’s
and buffer
systems for
multiple parallel
PCR systems.
3. Using the 04-2014 Test conducted on Black Spanish
protocol sample confirmed the presence
developed in 1 of GLRaV-1, GLRaV-2, GLRaV-3
and 2 test a and GFkV. Additionally showed
previously that the vine contained GLRaV-4,
characterised 04-2014 GLRaV-6, GVA, GVF and
infected vine RSPaV. Demonstrated
with multiple usefulness of the technique
virus infections.

4. Expand PCR 04-2017 The number of PCR systems for

systems for use in the protocol and their
use in the properties are listed in Table 1.
detection and These are theoretically capable of
identification of 04-2017 detecting 41 viruses of
grapevine grapevines
viruses within
the protocol.

5. Develop 04-2015 Analysis Pipeline provided in

methods of Figure 1. Detailed description of
data analysis of 06-2016 the development process
the Illumina provided in Progress reports
reads obtained 2015 and 2016.
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from parallel
NGS
sequencing of
the amplicons
of the PCR’s.

6. Assess the 04-2016 Multiplexing protocol established

multiplexing of along with positive/negative
samples using thresholds.
indexed pools 04-2016
of cloned
amplicons.

7. Using the PCR 04-2016 Test the optimised protocol using

protocol and nuclear material from Mr. T.
analysis Oosthuizen (Vititec) and Mrs. M.
pipeline Louw (Bosman Brothers).
developed test
the virus status 04-2016
of nuclear
plants from the
certification
scheme.

8. Using the PCR 04-2016 Test the optimised protocol using

protocol and mother-block material and
analysis samples with various symptoms
pipeline from mother-blocks (included
developed test table grape samples).
the virus status
of symptomatic 04-2017
vines from
foundation or
mother-block
vineyards.

9. Evaluate the 04-2013 Finalise laboratory detection of

routine GLRaV-3 and publish the
application of protocol. Field detection could not
GLRaV-3 be evaluated due to the
LAMP for field 04-2017 unavailability of sealed reaction
detection of vessels. We will attempt the use
GLRaV-3. of microcrystalline wax to seal the
reaction

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04-2013 LAMP shown to be more useful

10. Evaluate the than ELISA for the detection of
routine GLRaV-3 in five rootstocks types
application of
GLRaV-3 04-2013
LAMP for the
detection of
GLRaV-3 in
rootstocks
04-2013 Protocol shown to be useful for
11. Evaluate the the detection of South African
usefulness of a isolates aster yellows
published phytoplasma in both plants and
LAMP protocol 04-2013 leafhoppers
for the
detection of
Aster yellows
phytoplasma.

8. CONCLUSIONS

We have developed multiple PCR systems for the routine parallel, theoretical detection of 41
known viruses of grapevines. These PCR’s can be utilised directly within the certification
scheme, without consideration of the viral identity, as the primary aim is to have plants negative
for these viruses. By utilizing a subsequent, optimised, next generation sequencing analysis
pipeline the amplicon sequences derived from PCRs for any given sample can be pooled
together for the specific identification of viruses found. Furthermore, multiple samples can be
tested in parallel with this method utilising established positive/negative thresholds. This
protocol has been handed over for routine utilisation as a service to the grapevine industry to
the Virus Diagnostic unit of ARC-PPRI to provide this as a serve to the industry.

9. ACCUMULATED OUTPUTS

a) TECHNOLOGY DEVELOPED, PRODUCTS AND PATENTS

Develop genus wide, multi PCR systems for the parallel, theoretical detection of 41 known
viruses of grapevines. Develop a next generation sequencing pipeline for the analysis of
amplicon sequences derived from the amplicons for the identification of viruses found.

b) SUGGESTIONS FOR TECHNOLOGY TRANSFER

This protocol has been documented, and the protocol, primers and positive controls provided to
Ms. Marika van der Merwe, ARC-PPRI for the provision of the detection of these viral genera as
a service to the Wine Industry. Ms. E. Gagiano, SAPO, will also be trained in the use of this
protocol to use in a PhD study to detect viruses of table grapes.

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c) HUMAN RESOURCES DEVELOPMENT/TRAINING

Level of Total cost to

Student Name and Degree (e.g. studies in Graduation industry
Student Nationality MSc Agric,
Surname MComm)
final year of date throughout
project the project
Honours students
South African BSc.(Hons) MSc. 2013 R10000
J. Wayland
Microbiology (2013)
South African BSc(Hons) Hons 2014 R10000
Erika Bruck
Microbiology (2014)
South African BSc(Hons) Hons 2014 R10000
Kersitn Kenchenten
Microbiology (2014)
Masters Students
South African MSc. MSc. 2013 R36000
H. Walsh
Microbiology
South African MSc. MSc. 2017 R153300
J. Wayland
Microbiology
PhD students

Postdocs

Support Personnel

d) PUBLICATIONS (POPULAR, PRESS RELEASES, SEMI-SCIENTIFIC, SCIENTIFIC)

WALSH, H.A., AND PIETERSEN, G., 2013. Rapid detection of Grapevine leafroll-associated virus type
3 using a reverse transcription loop-mediated amplification method. Journal of Virological Methods
194: 308– 316

In Preparation: Development and optimization of a diagnostic system based on Illumina MiSeq

sequencing of genus wide PCR amplicons for the detection of viruses of grapevines
Jennifer Wayland, Anna E. C. Jooste and Gerhard Pietersen.

e) PRESENTATIONS/PAPERS DELIVERED

Wayland, J., Jooste, A.E.C., and Pietersen, G., 2015 The Optimization of a High-Throughput
Sequencing-based Diagnostic System for the Detection of Viruses of Grapevines. Virology Africa
2015, Cape Town, 30 November – 3 December 2015

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10. BUDGET

a) TOTAL COST SUMMARY OF THE PROJECT

YEAR CFPA DFTS Deciduous SATI Winetech THRIP OTHER TOTAL

2012 258300 258300

2013 274300 274300

2014 302000 112800 414800

2015 339124 169562 508686

2016 365948 365948

1539672 282362 1822034

b) FINAL BUDGET/FINANCIALS OF PROJECT

Proposed Actual cost

Project duration Variance Notes
budget incurred

TOTAL INCOME 1539672.00 1539672.00 0

Industry Funding

PHI Funding

Other Funding (THRIP) 282362 282362 0

TOTAL EXPENDITURE 1822034 1822034 0

Running Expenses

General operating costs 25220 25220 0

(printing, communication, etc.)

Local Travel 65350 65350 0

Publication costs

Lab Analysis

Lab Consumables 431589 431589 0

Other 0

Running expenses SUB- 522159 522159 0

TOTAL
HR Administration and Project
Management

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Proposed Actual cost

Project duration Variance Notes
budget incurred

HR Technical

HR Research 1080595 1080595 0

Student Bursaries 219280 219280 0

HR SUB-TOTAL 1299875 1299875 0

OTHER EXPENSES

Overheads

SURPLUS / DEFICIT 0 0 0

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EVALUATION BY INDUSTRY

This section is for office use only

Project number

Project name

Name of Sub-Committee*

Comments on project

Committee’s recommendation

• Accepted.

• Accepted provisionally if the sub-committee’s comments are also addressed.

Resubmit this final report by___________________________________

• Unacceptable. Must resubmit final report.

Chairperson__________________________________________ Date___________________

*SUB-COMMITTEES

Winetech
Viticulture: Cultivation; Soil Science; Plant Biotechnology; Plant Protection; Plant Improvement;
Oenology: Vinification Technology; Bottling, Packaging and Distribution; Environmental Impact; Brandy and Distilling;
Microbiology

Deciduous Fruit
Technical Advisory Committees: Post-Harvest; Crop Production; Crop Protection; Technology Transfer
Peer Work Groups: Post-Harvest; Horticulture; Soil Science; Breeding and Evaluation; Pathology; Entomology

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Figure 1: Schematic of genus-wide multiple PCR, Illumina read analysis for viruses of grapevine:

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Tabel 1: PCR systems and their properties developed for the routine detection of viruses of grapevines in the South
African wine Grape Certification Scheme.

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